On the Total Synthesis of Archaeal and Mycobacterial Natural Products
Holzheimer, Mira
DOI:
10.33612/diss.150711132
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Holzheimer, M. (2021). On the Total Synthesis of Archaeal and Mycobacterial Natural Products. University of Groningen. https://doi.org/10.33612/diss.150711132
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CHAPTER 5
Mycobacterium tuberculosis and its Cell
Wall Trehalose Glycolipids
This chapter introduces Mycobacterium tuberculosis and the disease tuberculosis. The mycobacterial cell envelope and its glycolipids and their role in Mycobacterium tuberculosis virulence and immunology is described.
162
Introduction – Mycobacterium tuberculosis
Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium belonging to the
mycobacteria genus. Mtb causes the disease tuberculosis (TB) in humans and is responsible for the highest number of deaths worldwide caused by a single bacterial pathogen with an annual death toll of nearly 1.5 million in 2018 as reported by the World Health Organization (WHO). It is estimated that a quarter of the global population is infected and therefore at risk of developing the disease TB. It was estimated that in 2018, approximately 10 million people developed the disease. In particular, Third World and developing countries are affected. Especially countries in Central and South-East Asia and Africa are battling an exceptionally high TB incidence (Fig. 1).1
Fig. 1 TB incidence estimated in 2018 for countries with more than 100.000 cases.1
Tuberculosis – disease, diagnosis and treatment
Mtb can be transmitted through the air from a patient with active TB to another
human by coughing or sneezing. The respiratory system is the first point of entry for Mtb. The bacterium is taken up by alveolar macrophages and can persist and replicate there until it ultimately kills these immune cells. Further bacterial replication eventually leads to the formation of granulomas in the lung. Most cases of TB proceed without symptoms (latent TB) and only approximately 10% of the patients develops symptoms and becomes infectious (open TB) towards others. The disease TB mainly occurs in the
The 30 high TB burden countries3 accounted for 87% of
all estimated incident cases worldwide, and eight of these countries accounted for two thirds of the global total: India (27%), China (9%), Indonesia (8%), the Philippines (6%), Pakistan (6%), Nigeria (4%), Bangladesh (4%) and
South Africa (3%) (Fig.3.3).
The severity of national TB epidemics in terms of the annual number of incident TB cases relative to popula-tion size (the incidence rate) varied widely among
coun-tries in 2018 (Fig.3.4 and Table3.4). There were under
10 incident cases per 100 000 population in most high- income countries, 150‒400 in most of the 30 high TB burden countries and above 500 in the Central African Republic, the Democratic People’s Republic of Korea, Lesotho, Mozambique, Namibia, the Philippines and South Africa. Among the 30 high TB burden countries, there were three with markedly lower incidence rates per capita: Brazil, China and the Russian Federation, which had best estimates of 45, 61 and 54, respectively.
An estimated 8.6% (range, 7.4‒10%) of the incident TB cases in 2018 were among people living with HIV (Table3.3 and Table3.4). The proportion of TB cases coinfected with HIV was highest in countries in the WHO African Region, exceeding 50% in parts of southern Afri-ca (Fig. 3.5). The risk of developing TB in the 37 million
3 These countries are listed in Table3.2, Table3.3 and
gated into six age groups (15–24, 25–34, 35–44, 45–54, 55–64 and ≥65 years) using data from national TB
prev-alence surveys implemented in 2007–2018 (Section3.4).
Country-specific distributions are used for countries that have implemented a survey; for other countries, the age distribution is predicted using prevalence survey data. Disaggregation by sex is based on actual M:F ratios for countries that have implemented surveys; for other coun-tries, this disaggregation is based on regional M:F ratios from a systematic review and meta-analysis (7). 3.1.2 Estimates of TB incidence in 2018 Globally in 2018, an estimated 10.0 million (range, 9.0–
11.1 million) people fell ill with TB,1 equivalent to 132
cas-es (range, 118–146) per 100 000 population. Estimatcas-es of
absolute numbers are shown in Table3.3 and estimates
of rates per capita are shown in Table3.4.
Most of the estimated number of cases in 2018 occurred in the WHO South-East Asia Region (44%), African Region (24%) and Western Pacific Region (18%); smaller propor-tions of cases occurred in the WHO Eastern Mediterra-nean Region (8.1%), Region of the Americas (2.9%) and
European Region (2.6%).2
1 Here and elsewhere in the report, “range” refers to the 95%
FIG. 3.3
Estimated TB incidence in 2018, for countries with at least 100 000 incident cases
South Africa Bangladesh Nigeria Pakistan Philippines Indonesia China India Number of incident cases 100 000 500 000 1 000 000 2 500 000
MYCOBACTERIUM TUBERCULOSIS AND ITS CELL WALL TREHALOSE GLYCOLIPIDS
lungs (pulmonary TB) and symptoms include chest pain, sputum producing cough (occasionally coughing blood), fever, weight loss and fatigue. The disease TB can also manifest outside of the lungs (extrapulmonary TB), for example in the lymphatic system, bones, central nervous system and other parts of the human body.2, 3
Fig. 2 Tuberculosis spectrum: from infection to active disease
(ã and reproduced with permission from MacMillan Publishers Limited).2
Currently, there are multiple methods applied in the diagnosis of Mtb to confirm infection of a patient (Fig. 2). A common method is sputum smear microscopy. Here, a sputum sample is treated with a dye and analysed under the microscope to detect tubercle bacilli which retain the staining agent, the so-called Ziehl-Neelsen staining. Additionally, a sputum sample can be cultured for various days, then treated with the stain and analyzed by microscopy, ensuring higher sensitivity than direct sputum analysis. Another common method to determine if an individual has been in contact with Mtb is the Mantoux tuberculin skin test (TST). Here, tuberculin (a glycerol extract from Mtb) is injected intradermally. After two to three days the point of injection is examined and if a skin reaction is visible, the individual likely has been or still is infected with Mtb and further tests are done to confirm. If no skin reaction is visible, the patient likely did not contract the disease. This skin test is known for its relatively high risk of false positive and negative results and is therefore not the method of choice in developed countries anymore. In developed countries, TB is also diagnosed by a blood test such as the QuantiFERON®–TB Gold In-Tube test or the T-SPOT®.TB test. Both blood
tests are commonly used in clinical diagnosis of TB in Europe and the United States of America, and both rely on the detection of peptidic antigens from
Mtb using an interferon-g release assay (IGRA). Besides these blood tests, a rapid diagnostic test has been developed, called Xpert MTB/RIF. This test allows detection of Mtb DNA sequences for diagnosis, notably not requiring blood samples but sputum samples. This enables fast and easy sampling and diagnosis. Despite multiple diagnostic tests for TB being available, research is focused on developing further rapid molecular-based diagnostic tests, as current tests are still not in the desired range of selectivity and sensitivity, thus occasionally producing false negative and false positive results.4-6
Additionally, the currently available tests are still too expensive for developing countries, thus not feasible for broad diagnosis in TB hotspots such as Africa, India, and South-East Asia. Consequently, there is a current state of ‘underdiagnosis’ in these regions, resulting in further spreading of the disease as well as preventing adequate and consequent treatment of infected individuals.1 Without treatment, TB has a high mortality rate of approximately
70% over 10 years (sputum positive patients, open TB). For smear-negative patients (closed TB), the mortality rate within 10 years of diagnosis
is estimated to be approximately 20%.7
If a patient has been diagnosed with TB (latent or active), there is a variety of small molecule drugs available for treatment. Currently, active drug-sensitive TB is treated by a 6-months regimen with four first-line antibiotics: isoniazid, rifampicin, pyrazinamide and ethambutol. In the first two months of this regimen, all four antibiotics are given to the patient and, in the remaining four months, only isoniazid and rifampicin are administered.8 The success rate of
this treatment is approximately 85%.1 This treatment is not without
restrictions, though, as patients can develop toxicity issues due to long treatment resulting mostly in liver and kidney problems among other side effects.9 For treatments to be successful, patient compliance is of paramount
importance to minimize the risk of relapse and the emergence of drug resistance.
For treatment of drug-resistant TB, second-line antibiotics such as aminoglycosides, fluoroquinolones, thioamides and oxazolidinones, are administered. These treatment regimens are characterized by high incidence of toxicity and other side effects and success rates are, at approximately 50%, significantly lower.1, 10
MYCOBACTERIUM TUBERCULOSIS AND ITS CELL WALL TREHALOSE GLYCOLIPIDS
In recent years, two new drugs (bedaquiline and delamanid) have been approved in the US and Europe for the treatment of multidrug-resistant TB (MDR-TB). These drugs are the first new drugs for TB to be marketed since approximately 40 years.11, 12 In addition, a lot of research on the development
of new drugs is ongoing and the current TB-drug pipeline is at an all-time high.13
The Holy Grail for the worldwide combat of Mtb infections though, is undoubtedly the development of a reliable and specific vaccine against Mtb.
The only licenced vaccine to date is the BCG vaccine.14 Efficacy of the BCG
vaccine in adults ranges between 0–80% (!), whereas in infants and children younger than five years the efficacy is around 50–80%.15, 16 Studies suggest
though that the protection by the BCG vaccine only last for around ten
years.17 Research is thus strongly focused on understanding the immunology
of Mtb to eventually be able to rationally develop a vaccine. Currently, there are various vaccine candidates under clinical testing.18, 19
The mycobacterial cell envelope
Mtb is characterized by a remarkably thick cell envelope, contributing up to
30% of its dried weight. The mycobacterial cell envelope is a complex, multi-layered barrier composed of various Mtb-specific (glyco-)lipids, oligo- and polysaccharides, and glycopeptides. Additionally, Mtb cells are coated with a complex mixture of polysaccharides, peptides and secreted lipids called the capsule (Fig. 3).20
The innermost part of the mycobacterial cell envelope is the plasma membrane (PM) composed of a lipid bilayer, and responsible for osmotic regulation. Next, there is a layer of highly cross-linked peptidoglycan (PG) which is built up by linear filaments of alternating N-acetylglucosamine and
N-acetylmuramic acid residues, which are cross-linked by peptides and
preserve the cell shape. The PG is covalently attached to the arabinogalactan (AG), a polymeric carbohydrate layer, which in turn is covalently attached to the mycolic acid layer by ester linkages. The mycolic acid layer – also called mycomembrane – is part of the mycobacterial outer layer. This outer layer is composed of two parts: the inner leaflet contains mostly mycolic acids, which are considered genus- and species-specific. The outer leaflet accommodates various mycobacterial glycolipids such as trehalose mono- and dimycolate
(TMM and TDM) and other trehalose-based glycolipids such as di-, tri- and pentaacyl trehaloses (DAT, TAT and PAT) as well as sulfolipids (SL). Furthermore, other glycolipids such as phenolic glycolipids (PGL), phthiocerol dimycocerosate (PDIM), lipoarabinomannan (LAM), and phosphatidylinositol
mannoside (PIM) can be found embedded in the outer mycomembrane.20-22
Lastly, the most outer part of the cell envelope is the surface-exposed capsule. It is a loose network mainly composed of polysaccharides and contains secreted lipids, proteases, lipases and porins. The capsule takes part in host-pathogen interactions, most importantly recognition by host phagocytes, which ultimately contributes to the survival and persistence of the bacterium in the host.23
MYCOBACTERIUM TUBERCULOSIS AND ITS CELL WALL TREHALOSE GLYCOLIPIDS
Fig. 3 Schematic representation of the molecular composition of the mycobacterial
cell envelope (ã and reproduced with permission from Elsevier).24
LM: lipomannan, OM: outer membrane, AG: arabinogalactan, PG: peptidoglycan, LAM: lipoarabinomannan, PM: plasma membrane, PIM: phosphatidylinositol
mannoside. O O O O O HO HO O O HO HO HOO HO O O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O O HO HO O O HO O O HO O OO O O OH O NH2 HO HO HO O O OHHO O O OH HO O OH O OHHO O O OH HO O OH O OHHO O OH O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO HO HO O O HO HO O O HO HO O O HO HO O O HO HO O HO HO O OH O HOHO OH O HN OH O OH O O O OH HO O OH O OHHO OH O O OH HO O OH O HO OH HO O O HO HO O O O O O O O HO HO O HO HO HOO HO O O O HO HO O O O HO HO O O HO HO O O O O HO HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O O HO HO O OH O HOHO O OH O HO HO O OH O HO HO HO O O HO HO O O HO HO O O O HO O HO HO O HO O HO HO O OH O HO HO O HO O HO HO O OH O HO HO O HO O HO HO O OH O HO HO O HO O HO HO O OH O HOHO O HO O HO HO O OH HO HO O O HO HO O OH O HOHO O OH O OHHO O OH O OHHO O OH O OH HO O OH O OHHO O O OHHO OH O O O O O HO HO O O HO HO O HOHO O O O HO HO O O O HO O HO O HO O O HO O OO HO O O HO HO O O HO O O HO HO O O HO O O O HO HO O O O HO O O HO O HO OH O O HO HO HO O HO OH O O HO HO OH O HO OH O O HO HOHO HO O O HO HO HO O O HO HO O O HO O O HO O O HO O OO HOO O O O HO HO OO HO O O O O HO HO O HO O O O O HO HO O HO O O O O HO HO O OH OH O O OH O OCH 3 OH O O O OH OH O OH O O CH 3 OH O O O O OH OH O O OH O OCH 3 OH O O O OH OH OH O O OH O O CH3 OO O OH O O OH OH OH O OH O OH HO OH O O OH O OOHOH O O HO O OH HO OH O O OH O O OH OH OH O OH O OH HO OH O OH O O OH O O OH OH OH O HO O OH HO OH O O HO O O OH OH OH O HOO OHHO OH O HOO O OH OH OH O HO O OH HO OH O O OH O O OH OH O O HO O OH HO OH O OH O O OH O O P O O O O OH OH OH O O OH OH OH OH O OH OH O OH OOH OH OHO O OH OH O O O OH OH O OO OH OH O O OH OH OH OH O OH OH OH OH OO OH OH O O O OH OH O O OH OH OH OH O O OH OH O O O OH OH O OOH OH OH OH O O OH OH O O O OH OH O O O OH OH O O OH OH OH O O OH OH OH OH OOH OHOH OH OH O OH OH OH O O OH OH OH O O OH OH OH OH O OH OH OH OH O OH OH OH OH OOH OH OH OH P H N H2N NHHN O O O O HN O HO NH NH2 HN O NHO O O O O NH2 O O O HO O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O O HO HO O O O O O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O O HO HO H N NHHN O O O O HN O HO O O N H NH2 HN O NHO O OOONH2 O NH O O OH OH NH O O O NH OH O O O OH OH N H O O O NH OH O O HO HO H N H2N NHHN O O O O HN O HO NH NH2 HN O N H O O O O O NH2 O O O O OH OH NH O O O NH OH O O O OH OH N H O O O NH OH O OH HO HO HN NH2 HN N HO O OO NH O OH NH NH2 NHNH O O O O H2N O H N O H N H2N NHNH2 O O O O HN O HO NH NH2 HN N H OO O ONH2 O O O O O HO O OH HONH OO O N H O O O OH OH N H O O O NH OH O O HO HO O O O O O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O O HO HO O O O O O OH OH N H O O O NH OH O O O OH OH N H O O O NH OH O O HO HO O O O O O OH OH N H O O O NH OH O O O OH OH N H O O O NH OH O OH HO HO HN H2N NHHN O O O O HN O HO NH NH2 HN O NHO O O O O NH2 O O O HO O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O O HO HO O O O O O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O O HO HO HN NHHN O O O O HN O HO O O NH NH2 HN O NHO O OOONH2 O NH O O OH OH NH O O O NH OH O O O OH OH N H O O O NH OH O O HO HO H N H2N NHHN O O O O HN O HO NH NH2 HN O NHO O O O O NH2 O O O O OH OH NH O O O NH OH O O O OH OH N H O O O NH OH O OH HO HO HN NH2 HN N HO O OO NH O OH NH NH2 NHNH O O O O H2N O H N O H N H2N NHNH2 O O O O HN O HO NH NH2 HN N H OO O ONH2 O HN NHHN O O O O HN O HO N H NH2 HN HNOOOONH2 O NH O HN H2N NHNH2 O O O O HN O HO NH NH2 HN NHOO O ONH2 O H N H2N NHHN O O O O HN O HO NH NH2 HN NHOO O O NH2 O HN NH2 HN N HO O OON H O OH NH NH2 NHNH O O O O H2N O HN O HN NHHN O O O O HN O HO N H NH2 HN NHOOOONH2 O NH O O O O O HO O OH OH NH O O O N H OH O O O OH OH NH O O O N H OH O O HO HO O O O O O OH OH N H O O O N H OH O O O OH OH NH O O O N H OH O O HO HO O O O O O OH OH NH O O O NH OH O O O OH OH N H O O O NH OH O O HO HO O O O O O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O OH HO HO O O O O O OH OH N H O O O N H OH O O O OH OH NH O O O N H OH O O HO HO O O O O O OH OH N H O O O NH OH O O O OH OH NH O O O N H OH O O HO HO O O O O O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O O HO HO O O O O O OH OH NH O O O N H OH O O O OH OH NH O O O NH OH O OH HO HO H N NHHN O O O O HN O HO N H NH2 HN HNOOOONH2 O NH O HN H2N NHNH2 O O O O HN O HO NH NH2 HN NHOO O ONH2 O HN H2N NHHN O O O O HN O HO NH NH2 HN NHOO O O NH2 O HN NH2 HN N HO O OON H O OH NH NH2 NHNH O O O O H2N O H N O H N NHHN O O O O HN O HO N H NH2 HN N H OOOONH2 O NH O HOO P NH3+ O O O P O O P HO OH O OHO O O HO O O P OO O O OH OH OH O O OH OH OH OH O OH OH OH OH P O O O O HO O P NH3+ O O O P NH3+ O O O P NH3+ O O O P NH3+ O O O P NH3+ O O O P NH3+ OO O P HO OH O O O P HO OH O O O P P NH3+ O O O P NH3+ O O O O P OO O O OH OH OH O O OH OH OH OH O OH OH OH OH P OO O OH OH OH OH OH O HOO HO O P NH3+ O O OP HO OH OO O P NH3+ OO O P NH3+ OO O P NH3+ O O OP HO OH OO O P NH3+ OO O P NH3+ O O OP HO OH O O O P NH3+ O O O P O O O O HO O P NH3+ O O O P NH3+ O O OP HO OH O O O P NH3+ O O O HO O P O O O OH OH OH OH OH OHO O P NH3+ O O O P NH3+ OO O P P O O O OH OH OH OH OH O P NH3+ O O O P NH3+ O O O P NH3+ O O O P NH3+ O O O P NH3+ O O O O O OHOH OH O OH OH OH OH O OH OH O OH O OH OHOH O O OH OH OH O O OH OHOH O O OH OH OH OH HOO HO O P NH3+ OO O P HO OH O O O O O OH OH OH O OH OH OH OH O OH OH O OH OOH OH OHO O OH OHO O O OH OHO O O OH OH O OOH OH OH OH OOH OH OH OH O O OH OH O O O OH OH O OOH OH OH OH O O OHOH O O O OH OH O OOH OH OH OH O O OH OH O O O OHOHO OO OH OH O O OH OH OH O OOH OH OH OH OH OO OH OH O O OH OH OH O O OH OH OH OH OOH OH OH OH OOH OH OH OH OOH OH OH OH O O HO O OH O OH O OH OH O OH OH O O O OH OH O O OH O O OH OH O OH OH O OO OH OH O OH OH O O O OHOH O OHOH O O OHOH O O O OH O O OH OHOO OH OH O OH OH O O OH OH O O O O OH OH OH O O OH OHOH OH O O O O O OH OH O O O OHOH O O OHOH O O OH OH O O OHOH O OH OH O O O OH OHOH O O OH OH OHO O OH OH OH OH O OH OH OO OH OH OO O OH O OHOHOO OH OH O OH OH O O OH OH O O O OH OH OH OH O O OH OH OH O O OH OHOH OH O OH OH OH O O OH OH OH OH O OH O OH O OH OH O OH OH O O O OH O H O OH O OOHOH O OH OH O O OOHOH O OH OH O O OOHO O O OH OH O O O OH OH O O OH OH O O OH OH O O OH OH OOHOH O O OOH OH OH O O OH OH OH O OOH OH OH OH OOHOH O O OH O H O OOHO O OH OH O OOHOH OOHOH O OOHOH O O OOH OH OH O O OH OH OH OH O H O O OH OH OH O O OH OH OH OH P NH3+ O O O P NH3+ O O O P NH3+ O O O P NH3+ O O O OH PM PG AG OM O P O O O O OHOH OH O O OH OH OH OH O OH OH O OH O OH OH OH O O OH OH O O OH OH OH O OH OH O O O O OH OH OH O OH HO OO OH O OOO O OH O O OH O O O O O O OH O O OH OH OH HOO OH HO O HO O O OO O OH O O OH O O O O OO PIM LAM Porin LM Mycolate O HO O OH OH OO O O OH OH OH O OH HO O O HO O O OH OH OO O OH OH OH O OH HO O HO OH P O O O HO OH P O O O O + NH2 H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N H2N NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2
FIGURE 2.1 A current perspective of the cell envelope of Mycobacterium tuberculosis. Represented is a theoretical model of the outer membrane (OM) wherein similar extractable lipids are present in both leaflets and the meromycolates of bound mycolic acids span the entire hydrophobic region. Another model proposes the meromycolate
Mycobacterial cell wall trehalose glycolipids
Trehalose glycolipids are major constituents of the mycobacterial cell envelope and there is vast structural diversity among members of this family. These glycolipids fulfil various functions: they act as virulence factors and effectors during host-pathogen interactions, serve as protection against the harsh intracellular environment (i.e. within host cells) and maintain cellular integrity. Besides contributing to overall pathogenicity, these cell envelope components are involved in cellular and humoral immune responses of the host.21, 22, 25-28 In the following section, the most prominent members of this
family – trehalose mycolates, sulfated glycolipids and acyl trehaloses – are described in detail.
Trehalose dimycolate
The most abundant of all glycolipids in the mycobacterial cell envelope is TDM (Fig. 4). TDM was first isolated in 1953 and its structure was proposed shortly thereafter in 1956. It was initially termed ‘cord factor’ as it was obtained from cording strains of Mycobacteria.29, 30 Later, it was shown that
also non-cording Mycobacteria produce TDM.31
TDM is comprised of a trehalose core esterified with two mycolic acids on the 6- and 6’-OH, whereas TMM (the biosynthetic precursor of TDM) only contains one mycolate moiety. The mycolic acids present in TDM and TMM are long fatty acid chains with a long alkyl branch in the a-position, and contain various functional groups such as cyclopropyl moieties, hydroxyl, keto and methoxy functional groups that serve as basis for the division into subgroups.32
MYCOBACTERIUM TUBERCULOSIS AND ITS CELL WALL TREHALOSE GLYCOLIPIDS
Fig. 4 Chemical structure of TDM.
TDM intercalates non-covalently with AG-linked mycolic acids and is crucial for maintaining cell wall integrity. This tight barrier formed of mycolic acids and TDM results in low antibiotic permeability and protection against an acidic environment.33 TDM has various biological functions, such as allowing Mtb to
survive within host phagosomes and promoting phagosome maturation arrest.34-36 TDM is involved in Mycobacterium-host recognition by direct
binding and activation of Mincle (macrophage-inducible C-type lectin)
resulting in macrophage activation as well as granuloma formation.37-39 TDM
is also highly antigenic and able to generate antibody responses of the host.40, 41 In recent years, synthetic TDM analogues are studied for potential
use as vaccine adjuvants.42-44
Sulfated trehalose glycolipids
Another class of trehalose-based mycobacterial glycolipids comprises the sulfated glycolipids, with sulfolipid-1 (SL-1)45-48 and its biosynthetic precursor
Ac2SGL (diacyl sulfoglycolipid) as major representatives.49, 50 These
glycolipids contain a trehalose core which carries a sulfate group at the 2’-OH and two to four fatty acid esters. The 2-2’-OH of trehalose carries a linear
O HO HO O OH OH OH O O HO O O OH OH O TDM
fatty acid chain (palmitic or stearic acid), whereas the 3’-OH, 6-OH and 6’-OH are esterified with long chain hepta- or octamethyl branched (in 1,3-arrays) phthioceranic and hydroxyphthioceranic acid (Fig. 5).
Fig. 5 Chemical structures of Ac2SGL and SL-1.
Studies with different Mtb mutants lacking both SL-1 and Ac2SGL or only
SL-1 and accumulating Ac2SGL, show that these sulfated glycolipids are not
required for Mtb growth.51-54 It has been suggested that SL-1 negatively
regulates intracellular Mtb growth54 and that the ratio of Ac2SGL to SL-1 might
be used by the bacterium to alter the host immune response to its advantage.55 To this date, the exact role of SL-1 and Ac
2SGL in Mtb virulence
remains unknown.
SL-1 has also been shown to inhibit macrophage and monocyte priming as
well as phagosome-lysosome fusion in cultured macrophages.56-59
Furthermore, SL-1 is highly immunogenic and has shown to be detectable by serodiagnostic tests.60, 61
Ac2SGL was first reported in 2004 and demonstrated to be a potent antigen
for CD1b-restricted T-cells.49 Since this makes it attractive for the
development of a subunit vaccine for Mtb, various synthetic analogues of Ac2SGL and other sulfated trehalose glycolipids have been prepared and
evaluated for their antigenic properties.62
The chemical structures including the absolute stereochemistry of both SL-1 and Ac2SGL ultimately have been confirmed by total synthesis.63, 64
O HO O O O OH OH O OH O HO Ac2SGL O O OH S O O ONa O HO O O O OH OH O O O O SL-1 O O OH S O O ONa O OH O OH
MYCOBACTERIUM TUBERCULOSIS AND ITS CELL WALL TREHALOSE GLYCOLIPIDS
Diacyl-, triacyl- and pentaacyl trehaloses
Fig. 6 Chemical structures of DAT1 and PAT.
The acyl trehalose family contains glycolipids which differ in the number and nature of their acyl moieties. These acyl trehaloses are known to come in series of alkyl chain-length homologues. To this date, three classes of these acyl trehalose glycolipids are known: diacyl- (DAT, 2,3-acyl),65, 66 triacyl-
(TAT, 2,3,6-acyl)67 and pentaacyl- (PAT, 2,3,6,2’,6’-acyl)68 trehaloses
(Fig. 6). A recent study has shown that the DAT family alone is comprised of more than 30 different molecular structures.69
Even though the acyl trehalose family has been subject of many studies, the ultimate role in Mtb pathogenesis remains unclear. Mtb mutants deficient of DAT, TAT and PAT show altered surface composition of the cell envelope resulting in an improved phagocytosis efficiency, albeit unchanged overall replication and survival of Mtb in in vivo mouse models. Thus, it was speculated that these acyl trehaloses serve the purpose to regulate the Mtb cell envelope composition by anchoring the outer capsule and therefore
preventing recognition and phagocytosis.70-72 Furthermore, DAT has shown
to inhibit the production of pro-inflammatory cytokines in monocytes indicating that DAT has the ability to negatively regulate the host immune response.73 DAT is not involved in receptor-mediated phagocytosis and
shows no cytotoxicity but is able to inhibit murine T-cell proliferation in vitro.74
DAT- and SL-deficient Mtb mutants are unable to block maturation, indicating
that these glycolipids may play and important role in Mtb pathogenesis.75
For many years, the acyl trehaloses received mainly attention due to their antigenicity and potential use as molecular markers for TB diagnosis, since it is known since their first isolation that Mtb infected patients produce specific antibodies against these glycolipids.60, 65, 67, 76-78 However, up to this date, no
O HO O O O OH OH O OH HO HO DAT1 O O O HO O O O OH OH O O O O PAT O O O O O
serodiagnostic test for Mtb infections based on anti-acyl trehalose antibodies could be developed due to great variation in sensitivity and specificity.79 The
acyl trehaloses DAT and TAT are also found to be agonists for the immune-receptor Mincle and thus are potential candidates for vaccine adjuvant design.44
To this date, members of the acyl trehalose family have escaped total synthesis, thus their exact molecular structure and stereochemistry is yet to be confirmed. In the following chapters, the first total synthesis of three members of the DAT family and the major component of the PAT family is described in order to prove or disprove the chemical structure and to provide synthetic reference material for further immunological studies.
Research aim
The chemical community has targeted a plethora of mycobacterial cell wall components including trehalose glycolipids for synthesis.63, 64, 80-86 Yet to this
date, two representatives of the trehalose glycolipid family have escaped total synthesis, namely DAT and PAT. These glycolipids are of great interest for immunological studies (vide supra) to better understand their role in host-pathogen interactions and their involvement in the host immune response. By chemical synthesis, sufficient amounts of synthetic reference material can be provided. This avoids tedious isolation from highly pathogenic, slow-growing
Mtb and, more importantly, the presence of minute amounts of biologically
active contaminants can be excluded. Furthermore, total synthesis of DAT and PAT will allow to prove or disprove the proposed chemical structures including stereochemistry by comparison with isolated natural material. The following chapters describe the total syntheses of both PAT and three members of DAT family as well as further immunological studies of the latter.
MYCOBACTERIUM TUBERCULOSIS AND ITS CELL WALL TREHALOSE GLYCOLIPIDS
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